Human natural killer (NK) cells exhibit a unique activation mechanism that is dominantly regulated by inhibitory receptors, including Killer Cell Inhibitory Receptors (KIR). KIR are the major NK cell receptors that bind classical major histocompatibility complex class I molecules (MHC-I) and transduce negative signals that restrain NK cell activation. A component of KIR negative signalling is recruitment of the protein tyrosine phosphatase SHP-1 to phosphorylated tyrosine residues within the receptor cytoplasmic domain. How SHP-1 functions to inhibit NK cell activation and whether additional signalling effector molecules also play roles in this process remain to be established. We have recently discovered that the protein tyrosine kinase (PTK), Csk, can also bind to tyrosine phosphorylated KIR. Csk recruitment by KIR provides an exciting basis for our future studies, since this PTK has the unique capacity to phosphorylate the regulatory tyrosine residues on receptor proximal PTKs of the Src family and terminate their activity. Such a novel negative signalling mechanism has not yet been identified for any inhibitory receptor, and we hypothesize that Csk recruitment provides KIR with capacity to dampen protein tyrosine phosphorylation-mediated activation events at an early stage. Additional data supports our hypothesis that phosphorylation status of the two individual tyrosine phosphorylation sites on KIR directly regulates effector enzyme binding profiles and functional outcomes. We propose to test our hypotheses with three specific aims: (1) to biochemically characterize Csk recruitment to KIR and its impact on PTKs of the Src family, (2) to determine the functional contributions of Csk recruitment on KIR inhibition of NK cell activation, and (3) to characterize the roles of the individual KIR tyrosines on inhibitory signalling responses. These studies will contribute substantially to our understanding of the mechanisms of KIR-mediated negative signalling. A better understanding of the mechanisms that dominantly regulate NK cell activation should ultimately allow for the development of therapeutic methods to beneficially manipulate their functions in therapies for cancer, viral infection, and prevention of tissue transplant rejection.